1. Field of the Invention
The present invention relates to a novel immortalized epithelial cell line containing a hybrid virus and method for its preparation. In particular the present invention relates to a hybrid virus derived from adenovirus 12 and simian virus 40 viruses which is then used for the immortalization of the epithelial cells.
2. Description of Related Art
Human cells are generally difficult to grow and maintain in long-term cultures in vitro. They have a limited life span in culture, grow for a short time and usually after 4 or 5 subcultures, they senescence and die.
Prostate cancer is the leading cancer in men in the United States, in terms of incidence. Thirty-two percent (32%) of all cancers in men arise in the prostate. It is estimated that 200,000 new cases of prostate cancer will occur in the U.S. in 1994. Prostate cancer is the second leading cause of death from cancer and 38,000 deaths are estimated to occur in 1994 (Boring et al., CA, Cancer J. Clin. 44:7, 1994). African American men have the highest incidence of prostate cancer in the world, which is almost twice as high as that in white men and more than 600 times higher than in men from Thailand (Webber et al., In Vitro Models for Cancer Res. Vol. V, pp. 3-24, Boca Raton, CRC Press, 1988). One in 10 men in the U.S. will develop prostate cancer in their lifetime (by age 85). An estimated 11 million men have latent or clinical prostatic carcinoma. Sixty-five percent (65%) of the cases already have metastatic disease at the time of diagnosis. The survival rate is less than 20%.
The causes of prostate cancer are not known at the present time. A study of the causes, prevention and treatment has been hampered by the fact that good animal or cell models are not available. Although rat prostatic cells have been used extensively for such studies, rat prostate is not homologous to the human prostate, thus, it is not an ideal system to use.
There is a need for cell lines derived from normal human prostate which can be used for studies on the process of prostate cancer development in man and to identify agents which may cause or prevent prostate cancer.
Attempts have been made to immortalize human adult prostatic epithelial cells using a monkey virus (Simian virus SV40; Cussenot, O., et al., Journal of Urology 143, 881-886 (1991); Kaighn, M. E., et al, Cancer Research, 49, 3050-3056 (1989); Lee et al., Internat. J. Oncol. 4, 821 (1994)).
Infection of mammalian cells with Simian virus 40 (SV40) can have two outcomes. In some simian cells which allow multiplication of the virus and are hence called "permissive cells", infection results in virus production and ultimately in cell death. Cells which do not permit virus multiplication are designated as "non-permissive" cells, such as rodent cells. Infection of these cells leads to induction and maintenance of an altered "immortalized" phenotype at a low frequency. Invariably, an integrated copy of viral DNA (vDNA) persists.
The viral DNA in non-permissive, SV40-immortalized cells usually cannot be induced to excise itself and replicate, but virus can be induced to replicate after fusion of non-permissive cells with permissive simian cells. According to Gish et al (Gish, W. R., et al., J. Virol., 61:2864-2876 (1987)), two essential viral components for this excision and lytic replication are cis-acting origin of replication (ori) and a replication component A gene product (large T antigen) acting in trans.
In culture, many types of human cells are semi-permissive for SV40 replication. The semi-permissive response of human cells to SV40 infection appears to be complex. Human cells can be productively infected by the virus, but they yield 100 times less virus than the simian cells. A few cells, which survive the lytic infection, may pass through a crisis period to yield immortalized cell lines which carry integrated copies of vDNA (Gish, W. R., et al., J. Virol., 61:2864-2876 (1987)). During this period, the rate of cell multiplication is markedly reduced or ceases. It has also been observed that cultures of SV40-infected human cells that were nursed through crisis no longer produced infectious SV40, although they had done so prior to crisis (Girardi, A. J., et al., J. Cell. Comp. Physiol. 65:69-84 (1965)). Furthermore, survivors of crisis exhibited higher levels of T-antigen staining than they did prior to crisis. High levels of T-antigen may be necessary for survival of cells through crisis (Girardi, A. J., et al., J. Cell. Comp. Physiol. 65:69-84 (1965)).
Three human cell lines immortalized only by SV40 virus alone have been developed in recent years. Immortalization of human neonatal prostate epithelial cells by strontium phosphate transection method using a plasmid (pRSV-T) containing SV40 was first accomplished by Kaighn et al., (Kaighn, M. E., et al., Cancer Res., 49:3050-3056 (1989)). The plasmid pRSV-T was developed at the National Cancer Institute. It is an SV40 ori construct containing the SV40 early region genes and the Rouse sarcoma virus long terminal repeat (Gorman, C., et al., Science, 221:551-553 (1983)). These cells formed rapidly growing, multi-layered colonies within two weeks and according to the authors, there was little or no indication of crisis. These cells contain cytokeratins and SV40-T antigen. They do not produce tumors in nude mice.
Cussenot et al (Cussenot, O., et al., J. Urol., 143:881-886 (1991)) used a plasmid containing SV40 genome with defective replication origin (ori-) encapsulated into liposomes. The cells were shown to contain the SV40 genome. They express large T-antigen and are positive for cytokeratins (CK) 18 and 19, weekly positive for prostatic acid phosphatase (PAP) and prostate specific antigen (PSA) and negative for CK 14. These cells contain high affinity receptors for 5.alpha.-dihydrotestosterone (5.alpha.-DHT).
In work similar to that of Cussenot et al., (J. Urol. 143:881-886, 1991), Lee et al., (Internat. J. Oncol. 4:821, 1994) used a plasmid (pRNS-1) containing an origin-defective SV40 genome and a plasmid carrying the neomycin resistance gene. A cell line was established, however, this cell line has not been characterized with regard to its prostatic epithelial origin on the basis of androgen responsiveness. These investigators were unsuccessful in establishing an immortalized prostatic epithelial cell line using Ad12-SV40 hybrid virus.
Adenoviruses have the ability to extend the lifespan of mammalian cells. When rat embryo cells are infected with adenovirus-2 (AD-2) virus, a fraction of the cells show extended lifespan but infectious virus could not be isolated from them although viral DNA persisted, as recognized by hybridization with labelled probes (Gallimore, P. H., et al., J. Mol. Biol. 89:49-72 (1974)).
In 1964, Rabson et al., (Proc. Soc. Exper. Biol. Med. 116:187-190, 1964), reported that the growth of adenoviruses in African green monkey kidney (AGMK) cell cultures is enhanced following pre-infection with SV40 virus. Schell et al (Schell, K., et al., Proc. Natl. Acad. Sci. USA, 55:81-88 (1966)) observed that the oncogenicity was markedly enhanced when adenovirus-12 (AD-12) and SV40 were passed serially together for five or more passages in AGMK cells. During the course of their investigation, Schell et al., (Schell, K., et al., Proc. Natl. Acad. Sci. USA, 55:81-88 (1966)) found that hybridization of the two viruses occurred. This hybrid virus was used in the present invention. This is the first successful immortalization of human prostatic epithelial cells with Ad12-SV40 hybrid virus.